Solenopsis invicta colonies were exposed to purified preparations of Solenopsis invicta virus 3 (SINV-3) to investigate virus pathogenesis at the colony level. Time course experiments revealed an infection exhibiting specificity for the adult stage (workers). SINV-3 genome and a capsid protein were increasingly present in worker ants with time. Northern blot analysis revealed two bands in RNA preparations from worker ants infected with SINV-3 corresponding to the genomic and sub-genomic species. Conversely, larval RNA preparations from SINV-3-infected colonies showed a near-complete absence of SINV-3 genome or sub-genome. The data confirm that SINV-3 is the etiological agent causing mortality among S. invicta colonies in the laboratory. We propose that SINV-3 infection somehow alters worker ant behavior, which may prevent them from acquiring and/or distributing solid food to the larvae. Consequently, larval mortality and impaired queen health occur as a result of starvation or neglect by the worker caste.